Bad Responders Research Articles

Effects of intermittent fasting on periodontal inflammation and subgingival microbiota.

Studies on the impact of intermittent fasting on periodontal health are still scarce. Thus, this study evaluated the effects of long-term intermittent fasting on periodontal health and the subgingival microbiota. This pilot study was part of a nonrandomized controlled trial. Overweight/obese participants (n=14) entered an intermittent fasting program, specifically the 5:2 diet, in which they restricted caloric intake to about a quarter of the normal total daily caloric expenditure for two nonconsecutive days/week. Subjects underwent a thorough clinical and laboratory examination, including an assessment of their periodontal condition, at baseline and 6months after starting the diet. Additionally, subgingival microbiota was assessed by 16S rRNA gene sequencing. After 6months of intermittent fasting, weight, body mass index, C-reactive protein, hemoglobin A1c (HbA1c), and the cholesterol profile improved significantly (p<0.05). Moreover, significant reductions were observed in bleeding on probing (p=0.01) and the presence of shallow periodontal pockets after fasting (p<0.001), while no significant change was seen in plaque index (p=0.14). While we did not observe significant changes in α- or β-diversity of the subgingival microbiota related to dietary intervention (p>0.05), significant differences were seen in the abundances of several taxa among individuals exhibiting ≥60% reduction (good responders) in probing pocket depth of 4-5mm compared to those with <60% reduction (bad responders). Intermittent fasting decreased systemic and periodontal inflammation. Although the subgingival microbiota was unaltered by this intervention, apparent taxonomic variability was observed between good and bad responders.

Abstract 222: Establishment of patient-derived tumor organoids from head and neck cancers for predicting response to treatments

Abstract The risk of relapse or recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is high despite the combination of surgery and radiochemotherapy, responsible for high toxicity. It is crucial to develop new therapeutic strategies and to identify patients likely to benefit from these treatments. Patient-Derived Tumor Organoids (PDTO) are three-dimensional multicellular structures derived from patient tumor samples and faithfully reproduce the histological and molecular characteristics of the original tumor. A growing body of research indicates that PDTO may predict the clinical response, representing a major opportunity for the development of new therapeutic strategies and precision medicine. The purpose of this translational study is to generate PDTO from HNSCC patients to evaluate their predictive value. PDTO were obtained after dissociation of tumor specimen of HNSCC patients through the setup of the clinical study ORGAVADS (NCT04261192). Tumor cells were embedded in extracellular matrix and cultured in specific medium. Histological and immunohistochemical characterizations were performed to validate the resemblance between PDTO and their original tumor. Response of PDTO to chemotherapy, radiotherapy and innovative therapies were evaluated by live-cell imaging and viability assays. The culture conditions were optimized to improve the success rate of PDTO establishment which now exceeded 50%. Twenty-one PDTO lines have been established and showed histological characteristics close to the original tumor. Expression of immunohistochemical tumor markers p53, p40, p63, and p16 were similar in tumors and paired PDTO. Functional assays were performed on 15 PDTO to analyze the response to treatments (cisplatin, olaparib, X-Rays) and heterogeneity of response was observed between the models. When clinical response was available, PDTO derived from good responders showed high sensitivity to treatments while PDTO from bad responders showed low sensitivity to treatments. Interestingly, two PDTO lines derived from HPV+ oropharyngeal tumors displayed very high sensitivity to cisplatin, matching with the patient's profile and response. Our results showed feasibility to derive PDTO from HNSCC and to perform functional assay to assess their response to different treatments. The first studies of correlation between PDTO and patient responses are promising and will be further investigated in more patients. This will allow to demonstrate the predictive value of PDTO from HNSCC with the purpose to develop a clinically compatible predictive functional assay. Citation Format: Marion Perréard, Vianney Bastit, Lucie Lecouflet, Guillaume Desmartin, Romane Florent, Corinne Jeanne, Juliette Thariat, Emmanuel Babin, Laurent Poulain, Louis-Bastien Weiswald. Establishment of patient-derived tumor organoids from head and neck cancers for predicting response to treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 222.

CXCL13 Positive Cells Localization Predict Response to Anti-PD-1/PD-L1 in Pulmonary Non-Small Cell Carcinoma.

Background: Immune checkpoint inhibitors (ICIs) have revolutionized non-small cell lung cancers (NSCLCs) treatment, but only 20-30% of patients benefit from these treatments. Currently, PD-L1 expression in tumor cells is the only clinically approved predictor of ICI response in lung cancer, but concerns arise due to its low negative and positive predictive value. Recent studies suggest that CXCL13+ T cells in the tumor microenvironment (TME) may be a good predictor of response. We aimed to assess if CXCL13+ cell localization within the TME can predict ICI response in advanced NSCLC patients. Methods: This retrospective study included 65 advanced NSCLC patients treated with Nivolumab/Pembrolizumab at IUCPQ or CHUM and for whom a pretreatment surgical specimen was available. Good responders were defined as having a complete radiologic response at 1 year, and bad responders were defined as showing cancer progression at 1 year. IHC staining for CXCL13 was carried out on a representative slide from a resection specimen, and CXCL13+ cell density was evaluated in tumor (T), invasive margin (IM), non-tumor (NT), and tertiary lymphoid structure (TLS) compartments. Cox models were used to analyze progression-free survival (PFS) and overall survival (OS) probability, while the Mann-Whitney test was used to compare CXCL13+ cell density between responders and non-responders. Results: We showed that CXCL13+ cell density localization within the TME is associated with ICI efficacy. An increased density of CXCL13+ cells across all compartments was associated with a poorer prognostic (OS; HR = 1.22; 95%CI = 1.04-1.42; p = 0.01, PFS; HR = 1.16; p = 0.02), or a better prognostic when colocalized within TLSs (PFS; HR = 0.84, p = 0.03). Conclusion: Our results support the role of CXCL13+ cells in advanced NSCLC patients, with favorable prognosis when localized within TLSs and unfavorable prognosis when present elsewhere. The concomitant proximity of CXCL13+ and CD20+ cells within TLSs may favor antigen presentation to T cells, thus enhancing the effect of PD-1/PD-L1 axis inhibition. Further validation is warranted to confirm the potential relevance of this biomarker in a clinical setting.

Abstract 3746: Dynamic characterization of small RNAs in non small cell lung cancer exosomes under immune-checkpoint inhibitor treatments

Abstract Immunotherapy based on Immune Checkpoint blockade (ICB) has become a significant therapeutic option for advanced Non Small Cell Lung Cancer (NSCLC) patients. However, there is an urgent need to find novel biomarkers that to reliably stratify good responders to immunotherapy. Currently, the available biomarkers are not specific enough. Exosomes are small membrane vesicles with sizes of 30-100 nm secreted by most cell types including cancer cells. Exosomes operate as an intercellular communication system by sending proteins, mRNA and miRNAs among other relevant RNA molecules. Exosomes enriched with miRNAS are involved in proliferation, differentiation, maduration and immune cell activation. Moreover, in cancer cells miRNA and other small RNA molecule expressions are dysregulated. Exosomes produced from cancer patient's plasma have been shown to be accurate diagnostic tools for the disease. In this study we profiled miRNAs and other small RNA cargo by exosome of plasma samples from 77 Non Small Cell Lung Cancer (NSCLC) metastatic patients before and after the first cycle of immunotherapy to evaluate the potentiality of predicting response to immunotherapy. We perform exosomes together with RNA isolation and small RNAseq sequencing from plasma samples before and after the first ICB cycle. Two independent softwares were used to identify small RNAs (RNAtoolbox and mirMaster). Prior ICB treatment we did not find differentially expressed miRNAs or other small RNA between good and bad responders. Interestingly we identified 12 exosomal miRNA differentially expressed between good and bad responders after the first cycle of ICB. Intriguingly, levels of miR-134-5p, miR-142-3p, miR-143-3p among others previously associated with NSCLC were found to be considerably higher in the good responder group than the bad responder group. Regarding other smallRNA molecules we observed a great variety and variability of piRNA, rRNA, scaRNA, lncRNA, snoRNA, snRNA, miscRNA and circRNA. To address the function of miRNA and other differentially expressed RNA molecules, we consulted KEGG, GO and Reactome for gene regulatory networks. Interestingly, KEGG results show pathways in cancer as top hit and Reactome highlight Immune System and cancer hits. In conclusion, we observed that patients that have favorable response to ICB have distinctive plasma exosomal miRNA patterns that could be used as possible biomarkers for predicting the effectiveness of immunotherapy in advanced NSCLC patients Citation Format: Maria Garrido-Barros, Javier Oliver, Juan Luis Onieva, Beatriz Martinez-Galvez, Jaime Duddelman, Antonio Rueda, Elisabeth Perez, Emilio Alba, Inmaculada Ramos, Juan Zafra, Manuel Cobo, Isabel Barragán. Dynamic characterization of small RNAs in non small cell lung cancer exosomes under immune-checkpoint inhibitor treatments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3746.

Risperidone response in patients with schizophrenia drives DNA methylation changes in immune and neuronal systems.

Background: The choice of efficient antipsychotic therapy for schizophrenia relies on a time-consuming trial-and-error approach, whereas the social and economic burdens of the disease call for faster alternatives. Material & methods: In a search for predictive biomarkers of antipsychotic response, blood methylomes of 28 patients were analyzed before and 4weeks into risperidone therapy. Results: Several CpGs exhibiting response-specific temporal dynamics were identified in otherwise temporally stable methylomes and noticeable global response-related differences wereobserved between good and bad responders. These were associated with genes involved in immunity, neurotransmission and neuronal development. Polymorphisms in many of these genes were previously linked with schizophrenia etiology and antipsychotic response. Conclusion: Antipsychotic response seems to be shaped by both stable and medication-induced methylation differences.

Analysis of the Variability in Different Criteria to Define the Success of Bariatric Surgery: Retrospective Study 5-Year Follow-Up after Sleeve Gastrectomy and Roux-en-Y Gastric Bypass.

(1) Background: The current criteria for defining good or bad responders to bariatric surgery based on the percentage of weight loss do not properly reflect the therapeutic impact of the main bariatric techniques. At present there is an urgent need to fill this gap and provide scientific evidence that better define the success or failure of bariatric surgery in the long term. (2) Methods: This is a retrospective database study of a prospective cohort with 5-year follow-up. We established the success or failure of bariatric surgery in terms of weight loss according to a selected criterion: (1) Halverson and Koehler; (2) Reinhold modified by Christou; (3) Biron; (4) TWL > 20%; (5) percentage of changeable weight (AWL > 35%). We analyzed sensitivity and specificity for successful weight loss. (3) Results: 223 (38.7%) patients underwent sleeve gastrectomy (LSG) and 353 (61.2%) underwent laparoscopic Roux-en-Y gastric bypass (LRYGBP). The success rates at 5 years are: EWL > 50% 464 (80%), Reinhold 436 (75.6%), Biron 530 (92%), TWL > 20% 493 (85.5%), AWL < 35% 419 (72.7); ≥50% EWL and alterable weight loss AWL > 35 were the most adequate criteria as their specificities and sensibility were far above >80%. (4) Conclusions: The present study shows how the different definitions of success or failure are inconsistent in relation to the outcomes of BS. However, there are some criteria that associate statistically significant differences for the resolution of comorbidities and show the highest sensitivity and specificity rates.

Abstract 3902: TRIM33 interacts with HSF1 in osteosarcoma development

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor in oncopediatrics. Despite therapeutic advances, the 5-year survival rate is 30% in bad responders to treatment or when pulmonary metastases are present at diagnosis. It is therefore urgent to better understand the molecular mechanisms involved in the development of OS in order to identify new therapeutic targets. As a major player in the stress response, it has recently been shown that the transcription factor HSF1 coordonates a stress-independent transcriptional program involving it in many pro-tumor biological processes in different cancers. A better understanding of the mechanisms by which HSF1 activity participates in tumor development is therefore essential to consider its therapeutic targeting. The co-partners of HSF1 to DNA, by modulating its transcriptional activity, could explain its pleiotropic effects. Based on this hypothesis, TRIM33 was identified by RIME. TRIM33 is known as a transcriptional cofactor and its involvement in various cancers. In this context, our objective is to study the interaction between TRIM33 and HSF1, their common genomic localization and to investigate the involvement of TRIM33 in the development of OS. Our results show that TRIM33 and HSF1 co-localize in the nucleus in OS TMA and by immuno-fluorescence in tumor cells and preclinical models. Furthermore, we observed by chIP-seq that TRIM33 and HSF1 share common DNA binding sites. Finally, our preliminary results show that TRIM33 invalidation by RNA interference in OS cell lines decreases tumor cell proliferation. Thus, in view of its role as a DNA partner of HSF1, TRIM33 is an interesting therapeutic target in OS. This work was supported by la Fédération Enfants et Santé, la SFCE et La ligue Contre le Cancer (comités 44, 49) Citation Format: Morgane Lallier, Robel Tefaye, Brice Moukengué, Céline Charrier, Rose-Anne Thepault, Benedicte Brounais-Le Royer, Marc Baud'huin, Franck Verrecchia, Benjamin Ory, François Lamoureux. TRIM33 interacts with HSF1 in osteosarcoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3902.

Metabolic Profile and Metabolite Analyses in Extreme Weight Responders to Gastric Bypass Surgery.

Background: Roux-en-Y gastric bypass (RYGB) surgery belongs to the most frequently performed surgical therapeutic strategies against adiposity and its comorbidities. However, outcome is limited in a substantial cohort of patients with inadequate primary weight loss or considerable weight regain. In this study, gut microbiota composition and systemically released metabolites were analyzed in a cohort of extreme weight responders after RYGB. Methods: Patients (n = 23) were categorized based on excess weight loss (EWL) at a minimum of two years after RYGB in a good responder (EWL 93 ± 4.3%) or a bad responder group (EWL 19.5 ± 13.3%) for evaluation of differences in metabolic outcome, eating behavior and gut microbiota taxonomy and metabolic activity. Results: Mean BMI was 47.2 ± 6.4 kg/m2 in the bad vs. 26.6 ± 1.2 kg/m2 in the good responder group (p = 0.0001). We found no difference in hunger and satiety sensation, in fasting or postprandial gut hormone release, or in gut microbiota composition between both groups. Differences in weight loss did not reflect in metabolic outcome after RYGB. While fecal and circulating metabolite analyses showed higher levels of propionate (p = 0.0001) in good and valerate (p = 0.04) in bad responders, respectively, conjugated primary and secondary bile acids were higher in good responders in the fasted (p = 0.03) and postprandial state (GCA, p = 0.02; GCDCA, p = 0.02; TCA, p = 0.01; TCDCA, p = 0.02; GDCA, p = 0.05; GUDCA, p = 0.04; TLCA, p = 0.04). Conclusions: Heterogenous weight loss response to RYGB surgery separates from patients’ metabolic outcome, and is linked to unique serum metabolite signatures post intervention. These findings suggest that the level of adiposity reduction alone is insufficient to assess the metabolic success of RYGB surgery, and that longitudinal metabolite profiling may eventually help us to identify markers that could predict individual adiposity response to surgery and guide patient selection and counseling.

Tumor Lymphocyte Infiltration Is Correlated with a Favorable Tumor Regression Grade after Neoadjuvant Treatment for Esophageal Adenocarcinoma.

(1) Background: We aimed to explore the association between neoadjuvant treatment, tumor-infiltrating immune lymphocyte (TIL), and tumor-associated macrophage (TAM) and survival in patients with esophageal adenocarcinoma. (2) Methods: Patients who underwent esophagectomy were divided into three groups according to their treatment modality and tumor regression grade (TRG): (i) surgery-only group (SG), (ii) good responders (GR) group (TRG 0–1), and (iii) bad responders (BR) group (TRG 2–3). We then carried out statistical correlations of the immunofluorescence analysis of the immune infiltrate in the esophageal surgical specimens with several clinical and pathological parameters. In addition, we analyzed The Cancer Genomic Atlas (TCGA) dataset for differences in TILs, TAMs, and protein expression in immune pathways. (3) Results: Forty-three patients (SG—15, GR—13, and BR—13) were evaluated. The highest enrichment of CD3+ (p < 0.001), CD8+ (p = 0.001) and CD4+ (p = 0.009) was observed in the stroma of GR patients. On multivariate analysis, only CD8+ T cell and signet-ring features were independent prognostic factors for overall survival. In TCGA analysis, we identified overexpression of TAM and colony-stimulating factor 1 receptor (CSF-1R). (4) Conclusions: High enrichment of lymphocyte subpopulations in the microenvironment of esophageal adenocarcinoma is associated with a favorable response to neoadjuvant treatment and an improved patient outcome.

KRAS gene mutation quantification in the resection or venous margins of pancreatic ductal adenocarcinoma is not predictive of disease recurrence

Pancreatic ductal adenocarcinoma (PDAC) patients eligible for curative surgery undergo unpredictable disease relapse. Even patients with a good pathological response after neoadjuvant treatment (NAT) remain susceptible to recurrent PDAC. Molecular analysis of R0 margins may identify patients with a worse prognosis. The molecular status of mutant KRAS (exon 2, codon 12/13) was analysed retrospectively by digital droplet PCR in tumour areas, venous and resection margins of resected tumours, either undergoing up-front surgery (UFS) or after NAT with a good pathological response. Expectedly, tumour tissues or remnants from patients who underwent NAT presented lower KRAS mutant allele frequencies (MAF) than patients eligible for UFS. Similarly, ypT1 tumour MAFs were greater than the ypT0 tumour remnant MAFs in the NAT group. Mutant KRAS status in margins did not distinguish NAT subgroups. It was not predictive of shorter recurrence-free or overall survival within or between groups. KRAS-double negativity in both venous and resection margins did not identify patients with a better prognosis, regardless of the groups. The cohorts ‘sizes were small due to limited numbers of patients meeting the inclusion criteria, but KRAS-positivity or MAFs in resection and venous margins did not carry prognostic value. Comparison of margins from good versus bad responders receiving NAT may provide better clinical value.

Ephrin-B2 PB-mononuclear cells reduce early post-stroke deficit in diabetic mice but not long-term memory impairment

Background and purposePost-stroke cognitive impairment (PSCI) has become a major public health issue, as a leading cause of dementia. The inflammation that develops soon after cerebral artery occlusion and may persist for weeks or months after stroke is a key component of PSCI. Our aim was to take advantage of the immunomodulatory properties of peripheral blood mononuclear cells (PB-MNC) stimulated with ephrin-B2/fc (PB-MNC+) for preventing PSCI. MethodsCortical infarct was induced by thermocoagulation of the middle cerebral artery in male diabetic mice (streptozotocin IP). PB-MNC were isolated from diabetic human donors, washed with recombinant ephrin-B2/Fc and injected into the mice intravenously on the following day. Infarct volume, sensorimotor deficit, cell death and immune cell densities were assessed on day 3. Six weeks later, cognitive assessment was performed using the Barnes maze. ResultsPB-MNC+ transplanted in post-stroke diabetic mice reduced the neurological deficit, infarct volume and apoptosis at D3, without modification of microglial cells, astrocytes and T-lymphocytes densities in the brain. Barnes maze assessment of memory showed that the learning, retention and reversal phases were not significantly modified by cell therapy. ConclusionsIntravenous PB-MNC+ administration the day after stroke induction in diabetic mice improved sensorimotor deficit and reduced infarct volume at the short term, but was unable to prevent long-term memory loss. To what extent diabetes impacts on cell therapy efficacy will have to be specifically investigated in the future. Including vascular risk factors systematically in preclinical studies of cell therapy will provide a comprehensive understanding of the mechanisms potentially limiting cell efficacy and also to identify good and bad responders, particularly in the long term.

Mechanisms of Resistance to Conventional Therapies for Osteosarcoma.

Simple SummaryOsteosarcoma (OS), the most common primary bone tumor, mainly affects children and adolescents. Unfortunately, in some cases, the absence of response to chemotherapy agents is observed, leading to metastases development and death of the patient. Resistance is one of the biological processes at the origin of therapeutic failure. In order to improve the therapeutic management of patients, it is necessary to identify and better understand the mechanisms underlying resistance. Here, we summarize molecular mechanisms of OS resistance to conventional chemotherapy and list some strategies that overcome resistance.Osteosarcoma (OS) is the most common primary bone tumor, mainly occurring in children and adolescents. Current standard therapy includes tumor resection associated with multidrug chemotherapy. However, patient survival has not evolved for the past decades. Since the 1970s, the 5-year survival rate is around 75% for patients with localized OS but dramatically drops to 20% for bad responders to chemotherapy or patients with metastases. Resistance is one of the biological processes at the origin of therapeutic failure. Therefore, it is necessary to better understand and decipher molecular mechanisms of resistance to conventional chemotherapy in order to develop new strategies and to adapt treatments for patients, thus improving the survival rate. This review will describe most of the molecular mechanisms involved in OS chemoresistance, such as a decrease in intracellular accumulation of drugs, inactivation of drugs, improved DNA repair, modulations of signaling pathways, resistance linked to autophagy, disruption in genes expression linked to the cell cycle, or even implication of the micro-environment. We will also give an overview of potential therapeutic strategies to circumvent resistance development.

Centromeric KIR AA Individuals Harbor Particular KIR Alleles Conferring Beneficial NK Cell Features with Implications in Haplo-Identical Hematopoietic Stem Cell Transplantation.

Simple SummaryWe have recently shown a broad disparity of Natural Killer (NK) cell responses against leukemia, highlighting good and bad responders resting on the Killer cell Immunoglobulin-like Receptors (KIR) and HLA genetics. In this study, we deeply investigated KIR2DL NK cell repertoire in combining high-resolution KIR allele typing and multicolor flow cytometry from a cohort of 108 blood donors. Our data suggest that centromeric (cen) AA individuals display more efficient KIR2DL alleles (L1*003 and L3*001) to mount a consistent frequency of KIR2DL+ NK cells and to confer an effective NK cell responsiveness. The transposition of our in vitro observations in T-replete haplo-identical Hematopoietic Stem Cell Transplantation (HSCT) context led us to observe that cenAA HSC grafts limit significantly the incidence of relapse in patients with myeloid diseases after T-replete haplo-identical HSCT. As NK cells are crucial in HSCT reconstitution, one could expect that the consideration of KIR2DL1/2/3 allelic polymorphism could help to refine scores used for HSC donor selection.We have recently shown a broad disparity of Natural Killer (NK) cell responses against leukemia highlighting good and bad responders resting on the Killer cell Immunoglobulin-like Receptors (KIR) and HLA genetics. In this study, we deeply studied KIR2D allele expression, HLA-C recognition and functional effect on NK cells in 108 blood donors in combining high-resolution KIR allele typing and multicolor flow cytometry. The KIR2DL1*003 allotype is associated with centromeric (cen) AA motif and confers the highest NK cell frequency, expression level and strength of KIR/HLA-C interactions compared to the KIR2DL1*002 and KIR2DL1*004 allotypes respectively associated with cenAB and BB motifs. KIR2DL2*001 and *003 allotypes negatively affect the frequency of KIR2DL1+ and KIR2DL3+ NK cells. Altogether, our data suggest that cenAA individuals display more efficient KIR2DL alleles (L1*003 and L3*001) to mount a consistent frequency of KIR2DL+ NK cells and to confer an effective NK cell responsiveness. The transposition of our in vitro observations in the T-replete haplo-identical HSCT context led us to observe that cenAA HSC grafts limit significantly the incidence of relapse in patients with myeloid diseases after T-replete haplo-identical HSCT. As NK cells are crucial in HSCT reconstitution, one could expect that the consideration of KIR2DL1/2/3 allelic polymorphism could help to refine scores used for HSC donor selection.

Prognostic value of the early change in neutrophil-to-lymphocyte ratio in metastatic pancreatic adenocarcinoma

In metastatic pancreatic adenocarcinoma, a high neutrophil-to-lymphocyte ratio (NLR) at diagnosis is a marker of poor prognosis. The prognostic role of baseline NLR and NLR change during first-line chemotherapy were determined. We conducted a retrospective study by using data from a single-center prospective cohort and a randomized open-label, multicenter, randomized trial. Two hundred and twelve patients were analyzed. Baseline NLR>5 was an independent marker of poor prognosis for overall survival (HR=2.01, 95% CI 1.33−3.05; P=0.001) and progression-free survival (PFS; HR=1.80, 95% CI 1.23–2.65; P=0.0026). According to NLR dynamics (n=172), patients with NLR≤5 on days 1 and 15 had a significantly better prognosis than those with NLR≤5 on day 1 and NLR>5 on day 15 (HR=2.23, 95% CI 1.18–4.21; P=0.013), NLR >5 on day 1 and NLR ≤5 on day 15 (HR=3.25, 95% CI 1.86–5.68; P<0.001), and NLR>5 on days 1 and 15 (HR=3.37, 95% CI 1.93–5.90; P<0.001). Over time, bad responders (PFS <6 months) had significantly higher mean NLR than good responders (PFS>6 months; group effect: P<0.0001). Seven out of eight patients with baseline NLR>5 had circulating tumor DNA. This study confirmed the independent prognostic value of baseline NLR >5 in metastatic pancreatic cancer. The change in NLR early during chemotherapy was also a prognostic indicator in patients with NLR ≤5.

Abstract 281: Evaluation of circular RNA profiling in metastatic melanoma patients treated with immune checkpoint blockade

Abstract Melanoma comprises a variety of malignant cell types from the skin, the mucous membranes, and the pigmented cells of the eye. Its incidence and mortality rates continue to rise worldwide, mainly driven by increased ultraviolet light exposure. Although screening improvements have led to a better diagnosis in the early stages, the outcome for patients with Metastatic Melanoma (MM) remains poor and the survival rate is considerably low. Recently, Immune Checkpoint Inhibitors (ICB) have revolutionized the treatment of MM, although only 30% of patients enjoy clinical benefit and a plethora of different molecular mechanisms is postulated to cause resistance. Several biomarkers are currently on the spotlight as putative predictors of response; however, they are not sufficiently informative and robust. Circular RNAs (circRNAs) are a class of single-stranded stable RNAs produced by 5′-to-3′ transcription of coding gene exons or long non-coding RNAs that act as translation templates, RNA-binding protein regulators, or miRNA-binding sponges. Here, we present an exploratory study to evaluate the potential use of circRNA as ICB response predictors. We recruited 23 MM patients treated with Nivolumab and categorized them as good responders (10 patients) and bad responders (11 patients). All RNA-seq with ribosomal depletion was performed on RNA isolated from pre-treatment FFPE tumor biopsies. Two bioinformatic tools that use de novo assembly for the identification of circRNAs (Starchip and Ciri) were employed independently to characterize the MM circRNAs profile and to identify a specific pattern for responders. Overall, a total of 731 circRNAs were detected. Interestingly, the number of circRNAs were comparatively higher in bad responders (11.917 reads, 321 circRNAs) versus good responders (7368 reads, 176 circRNAs), with 234 common circRNAs. Additionally, we also explored the biological function of the identified circRNAs in MM, by interrogating KEGG, GO and Reactome databases to delineate the gene regulatory networks specifically associated with the response-related phenotypes. The circRNAs exclusively expressed in patients presenting resistance to Nivolumab were associated with Transcriptional Misregulation in Cancer and Viral Infections processes. In contrast, circRNAs expressed uniquely in good responders were enriched in Generic Transcription pathways. Importantly, amongst the identified circRNAs, CDR1-AS was differentially downregulated in good responders (536 vs 1580 reads, p-value 0.002). Remarkably, CDR1-AS acts as miRNA-binding sponge of miRNA-7 in the context of invasion and migration in melanoma. In conclusion, our exploratory study paves the way to the utilization of novel biomarkers associated to immunotherapy failure in MM patients. Moreover, our results suggest that CDR1-AS is a novel putative predictor of ICB response. Citation Format: Juan Luis Onieva, Javier Oliver, Aurora Laborda-Illanes, Maria Rosario Chica-Parrado, Alicia Garrido-Aranda, Cynthia Robles-Podadera, Daniel Prieto, Elena Gallego, Alfonso Sanchez, Iñaki Comino, Vanessa De Luque, Martina Alvarez, Maria Jose Lozano, Pedro Jimenez, Miguel Angel Berciano-Guerrero, Emilio Alba, Manuel Cobo, Isabel Barragan. Evaluation of circular RNA profiling in metastatic melanoma patients treated with immune checkpoint blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 281.

Genetic and Molecular Basis of Heterogeneous NK Cell Responses against Acute Leukemia

Natural killer (NK) cells are key cytotoxic effectors against malignant cells. Polygenic and polymorphic Killer cell Immunoglobulin-like Receptor (KIR) and HLA genes participate in the structural and functional formation of the NK cell repertoire. In this study, we extensively investigated the anti-leukemic potential of NK cell subsets, taking into account these genetic parameters and cytomegalovirus (CMV) status. Hierarchical clustering analysis of NK cell subsets based on NKG2A, KIR, CD57 and NKG2C markers from 68 blood donors identified donor clusters characterized by a specific phenotypic NK cell repertoire linked to a particular immunogenetic KIR and HLA profile and CMV status. On the functional side, acute lymphoblastic leukemia (ALL) was better recognized by NK cells than acute myeloid leukemia (AML). However, a broad inter-individual disparity of NK cell responses exists against the same leukemic target, highlighting bad and good NK responders. The most effective NK cell subsets against different ALLs expressed NKG2A and represented the most frequent subset in the NK cell repertoire. In contrast, minority CD57+ or/and KIR+ NK cell subsets were more efficient against AML. Overall, our data may help to optimize the selection of hematopoietic stem cell donors on the basis of immunogenetic KIR/HLA for ALL patients and identify the best NK cell candidates in immunotherapy for AML.

Self-Regulation of SMR Power Led to an Enhancement of Functional Connectivity of Somatomotor Cortices in Fibromyalgia Patients.

Neuroimaging studies have demonstrated that altered activity in somatosensory and motor cortices play a key role in pain chronification. Neurofeedback training of sensorimotor rhythm (SMR) is a tool which allow individuals to self-modulate their brain activity and to produce significant changes over somatomotor brain areas. Several studies have further shown that neurofeedback training may reduce pain and other pain-related symptoms in chronic pain patients. The goal of the present study was to analyze changes in SMR power and brain functional connectivity of the somatosensory and motor cortices elicited by neurofeedback task designed to both synchronize and desynchronize the SMR power over motor and somatosensory areas in fibromyalgia patients. Seventeen patients were randomly assigned to the SMR training (n = 9) or to a sham protocol (n = 8). All participants were trained during 6 sessions, and fMRI and EEG power elicited by synchronization and desynchronization trials were analyzed. In the SMR training group, four patients achieved the objective of SMR modulation in more than 70% of the trials from the second training session (good responders), while five patients performed the task at the chance level (bad responders). Good responders to the neurofeedback training significantly reduced pain and increased both SMR power modulation and functional connectivity of motor and somatosensory related areas during the last neurofeedback training session, whereas no changes in brain activity or pain were observed in bad responders or participants in the sham group. In addition, we observed that good responders were characterized by reduced impact of fibromyalgia and pain symptoms, as well as by increased levels of health-related quality of life during the pre-training sessions. In summary, the present study revealed that neurofeedback training of SMR elicited significant brain changes in somatomotor areas leading to a significant reduction of pain in fibromyalgia patients. In this sense, our research provide evidence that neurofeedback training is a promising tool for a better understanding of brain mechanisms involved in pain chronification.

Muramyl Tripeptide Plus Chemotherapy Reduces Metastasis in Non-Metastatic Osteosarcoma: A Single-Center Experience.

Background:The immunomodulator mifamurtide plus a chemotherapy regimen has been shown to significantly improve the outcome in non-metastatic osteosarcoma patients. We report the results of the addition of mifamurtide to chemotherapy in newly diagnosed patients with osteosarcoma. Methods:A total of 36 children with osteosarcoma without detectable metastasis were treated between November 2010 and April 2018 at the Ankara University Department of Pediatric Oncology. Mifamurtide was added to the chemotherapy regimen in 17 patients while the remaining 19 did not receive mifamurtide. The probabilities of metastasis and overall survival were compared between the groups. Results:The 43-month survival rate was 87.5% and 89.9% in the patients who received and did not receive mifamurtide, respectively (p=0.65). Common side effects of mifamurtide were chills and fever. The addition of mifamurtide in the high-risk group with ≤95% necrosis tended to decrease the probability of distant metastasis (36.4% vs. 58.3%) (p=0.39). The time to metastasis in the group with positive surgical margins (4 months in one patient in the non-mifamurtide group, 7 and 20 months in the mifamurtide group) was also longer in the mifamurtide group. During the 43-month follow up period, median time to metastasis was longer in the mifamurtide group (20 vs. 5 months). In addition, mifamurtide plus chemotherapy decreased the risk of metastasis in the cases with primary site relapse. Conclusions:The addition of mifamurtide to chemotherapy might improve event-free survival by decreasing the probability of distant metastasis in bad histologic responders, and also by increasing the time to distant metastasis in the surgical margin positive group. Additional clinical studies are necessary to determine the long-term effects of mifamurtide on metastatic disease.

Evaluation of growth hormone co-treatment in in vitro fertilization in patients responding better to the GnRH antagonist short protocol.

ObjectiveThe present study aims at evaluating the results obtained after in vitro fertilization in bad responders, using controlled ovarian hyperstimulation together with the use of gonadotrophin releasing hormone (GnRH) antagonist (cetrorelix acetate) in a short protocol.MethodsThis is an analytical, longitudinal, retrospective and controlled study involving patients who underwent in vitro fertilization (IVF) procedures in the assisted reproduction program of the Reproferty clinic, in the municipality of São José dos Campos/SP, from January 2012 to December 2016. We collected the data obtained from the medical records of patients considered to have undergone controlled ovarian hyperstimulation using GnRH antagonist (cetrorelix acetate) and Growth Hormone (GH) in a short cycle protocol. The patients considered controls were those submitted to the same hyperstimulation process, without using GH.ResultsThere were significant differences in the following analyzed parameters: gonadotrophin regimen dose, stimulation duration, and estradiol levels on the day of HCG administration, number of follicles, number of retrieved oocytes, number of mature oocytes and number of good-quality embryos. On the other hand, the GH administration was not significant in the number of cycles that achieved transfer, the number of embryos transferred and the number of frozen cycles. In the case group, there was no increase in the number of cycles that reached pregnancy rate βhCG+; however, the clinical pregnancy rates and live birth rates were significant.ConclusionThe present investigation demonstrated that GH administration as a supplement in poor responders improves the majority of the parameters to achieve a full term pregnancy in these patients.

Preferential Activity of Petiveria alliacea Extract on Primary Myeloid Leukemic Blast.

The need for new therapeutic approaches to improve the response in acute leukemia (AL), either by directing therapy or with new therapeutic alternatives, has been a research and clinical interest topic. We evaluated whether blasts from AL patients were sensitive ex vivo to the induction chemotherapy and whether the extracts of Petiveria alliacea (Anamu SC) and Caesalpinia spinosa (P2Et) modulated the sensitivity of leukemic cells to death. Bone marrow samples were taken from 26 patients with de novo AL and 6 in relapse, and the cytotoxicity of the extracts alone or in combination with the chemotherapeutic was evaluated by XTT. Patients were classified as good (GR) and bad responders (BR) according to the ex vivo test. 70.5% of the GR patients to the ex vivo test achieved postinduction remission to induction chemotherapy with a median overall survival of 12.50 months versus 7.23 months in the two groups. Furthermore, it was found that the ex vivo response to extracts and chemotherapeutics is heterogeneous and shows an exclusive pattern between the extracts, Anamu being the more effective in inducing cell death. The combination of extracts with chemotherapeutic agents showed synergistic or antagonistic effects in the patients' blasts. These results show that the ex vivo evaluation of the sensitivity to induction drugs using primary blasts from patients exhibits a correlation with the response to induction chemotherapy in patients. These analyses would allow establishing a system to predict response to treatment and determine ex vivo susceptibility to new therapies under development, among which is phytotherapeutics.